The recognition of viral peptides in the context of MHCI proteins by cytotoxic T lymphocytes is a key event for eliminating virus-infected cells. Prior to presenting peptides at the cell surface, MHCI proteins must undergo a stringent maturation process in the lumen of the endoplasmic reticulum by transiently interacting with members of the peptide loading complex (PLC). After dissociation from the PLC, peptide-bound MHCI proteins transit through the Golgi to the surface following the secretory pathway. The secretory pathway is known to involve a series of membrane bound compartments with specialized functions in cargo transport; however, many of the molecular details of this pathway have yet to be elucidated. To resolve outstanding questions of MHCI biosynthesis along the secretory pathway, we will use viral immune evasion proteins as probes for physiologic pathways. Viruses encode a plethora of immune evasion proteins that exploit a surprising diversity of physiologic pathways to block antigen presentation and they do so with great specificity and potency. These properties make immune evasion proteins highly effective probes for defining molecular pathways of protein quality control that are of particular relevance to MHCI function. In this grant, we will use immune evasion proteins to probe i) how misfolded MHCI proteins are identified and translocated from the ER lumen to the cytosol, ii) mechanisms of ER to Golgi recycling and their importance for MHC quality control and initiating ERAD, and iii) the physiologic role of ubiquitination of lysine vs. serine MHCI residues for ERAD and recycling from the plasma membrane. The proposed experiments will use cell biological, biochemical, and structural approaches employed by collaborative efforts of the Hansen and Fremont labs.